Title

Author

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor(s)

Dr. Brian Shilton

Abstract

Quinone reductase 2 (NQO2) is a mammalian enzyme that catalyzes the reduction of quinone using an unusual co-substrate, dihydronicotinamide riboside (NRH). In addition, NQO2 has a secondary function, which is to modulate the 20S proteasomal degradation of p53 in a redox-dependent manner. This alternate function has been characterized in its sister enzyme quinone reductase 1 (NQO1) and yeast quinone reductase Lot6p, but relatively few studies have investigated the role of NQO2 beyond quinone catalysis. From studies of Lot6p, it was proposed that quinone reductases could be categorized as flavin redox switches. In this thesis, the how NQO2 functions as a flavin redox switch and how inhibitors can modulate NQO2 was investigated. Initially, a method was developed to reconstitute recombinant NQO2 with FAD. Having purified functional NQO2, reduced structures of NQO2 were solved in complex with four different inhibitors: the antimalarial drug chloroquine, CK2 inhibitor DMAT, DNA intercalating agent ethidium bromide, and acridine orange. In all four reduced structures, the hydrogen bond network and the orientation of inhibitors in the active site of NQO2 were changed by the presence of water molecules compared to the oxidized structures. For the structure of reduced NQO2 in complex with chloroquine, there was also a global conformational change. These reduced structures together showed that NQO2 has two functional states, and indicated mechanistic features of a flavin redox switch in NQO2. To understand how these inhibitors affect NQO2 signalling in cells, the CK2 inhibitors and DNA intercalating agents were further studied in tissue culture. Even though the cytotoxicity of these compounds was not dependent on NQO2, p53 levels were modulated in an NQO2-dependent manner. In conclusion, this thesis has shown that in addition to quinone catalysis, NQO2 functions as a flavin redox switch, and NQO2 inhibitors have the ability to modulate this process.